Myosin is the muscle protein primarily responsible for the coupling of the chemical energy of ATP to the production of mechanical force and work. It is one of the few energy transducing ATPases which can be obtained in large quantities and in a relatively low molecular weight, water soluble form. As such it serves as an excellent model system for studying energy transduction on a molecular level. The goal of the work proposed here is to use NMR to define and characterize, both structurally and thermodynamically, the two macromolecular states of myosin which have been recently described (Shrive, J. and Sykes, B.D. Biochemistry 20:2004-2012). Strong evidence has been presented indicating that myosin can exist in two fundamental, discrete states, MR and MT, both of which can hydrolyze ATP. The present data indicates that a transition from MR to MT is expressed as the power stroke when myosin is bound to actin. The nature of the difference between these two states and Delta H degrees and Delta S degrees will be determined using 31P, 19F, and 1H NMR. 31P NMR studies will be performed with probes bound in the active site, e.g. AMP.PNP, ADP, and IDP. 19F NMR studies will be carried out using a fluorine-containing probe specifically attached to sulfhydryl-1. A 500 MHz 1H NMR study will be used to determine the extent of the conformational differences between the two states, and to investigate the existence of similar states in the absence of nucleotides. The effect of calcium and the DTNB light chain on the states and their relative energies will be investigated. In addition 31P NMR studies will be performed on "trapped" nucleotides. These results will be correlated with data from fluorescence and UV difference spectroscopy and steady state kinetics. The relationship between the sharp breaks in ATPase and ITPase Arrhenius plots and the relative energies of the two states as a function of temperature will be investigated. The existence and relative energies of two discrete states of the cross-bridge bound to actin which differ in angle of attachment will be studied in oriented muscle fibers using 19F-labeled cross-bridges. This work has important implications for the fields of muscle control and muscle disease since alterations in the energies and barriers between the R and T states will result in alterations in the energy coupling efficiency of muscle.